We have focused our efforts on studying hemoglobin switching of human beta-like globin genes in the erythroleukemia cell line K562. The absence of beta globin gene expression in K562 cells appears to be related to environmental regulatory factors, which may involved in the presence of the silencer (e.g. BP-1 and/or BP-2) and absence of activator (e.g. Erythroid Krhpple-Like Factor (EKLF) transcription factors or both. Structural analysis, including gel mobility shift and DNAse I hypersensitive footprinting, techniques, revealed the 5= upstream regulatory region of the beta-globin gene contains three structural motifs (-530, -304, -289) that are bound by two putative trans-acting factors, termed beta-protein 1 and beta-protein 2 (BP1, BP2) (Berg, P. et al, N.A.R., 17:8833, 1989). In situ mutagenesis of each structural motif introduced into a pCAT reporter vector followed by transient transfection into hemin-induced K562 cells yielded levels of CAT activity as much as 5-fold (ranging from 49% to 69% CAT induction, p<0.001) composed to wt beta CAT and therefore further confirmed these distal promoter sequences as silencers of beta-globin expression. Mutagenesis of all three cis-acting elements, however; resulted in CAT activity lower than wt beta CAT (5% versus 13%, p<0.05). Sequence analysis revealed the beta-globin motifs for BP1 and BP2 have overlapping binding sites with the chromosomal protein high mobility group protein 1 (HMG1), a non-specific DNA-binding and bending factor. We theorized that the removal of BP1 and BP2 binding sites also disturbed the binding of HMG1, thus impede DNA-protein and/or protein-protein interactions needed to facilitate gene expression. In order to further characterize the tertiary complex between the beta-globin cis-acting elements with BP1/BP2 and HMG1, chimeric constructs have also been created. Placing proximal BP1 and BP2 binding sites in either a cis- or trans- position to each other (by inserting 1/2 or 1 full turn of DNA, respectively) does not markedly affect beta-globin silencing, suggesting the silencing function does not require proximal BP1-BP2 interaction. However, inserting an additional HMG1-site between these protein binding sites caused a 4-fold CAT induction, suggesting that this disturbance of the architectural formation impedes beta-globin silencing. Through the study of the combinatorial DNA cis-acting elements and trans-acting factors, a likely molecular mechanism for the developmental silencing of the human adult beta-globin gene is evolving.